Novel radiopharmaceuticals and chelating agents useful in their preparation

ABSTRACT

A dihydropyridine←→ pyridinium salt type of redox, or chemical, delivery system for the site-specific and/or site-enhanced delivery of a radionuclide to the brain is provided. A chelating agent capable of chelating with a radionuclide and having a primary, secondary or tertiary amino function can be converted to the corresponding analogue in which said function is replaced with a dihydropyridine← → pyridinium salt redox system and then complexed with a radionuclide to provide a new radiopharmaceutical that, in its lipoidal dihydropyridine form, penetrates the blood-brain barrier (&#34;BBB&#34;) and allows increased levels of radionuclide concentration in the brain, particularly since oxidation of the dihydropyridine moiety in vivo to the ionic pyridinium salt retards elimination from the brain while elimination from the general circulation is accelerated. 
     This radionuclide delivery system is well suited for use in scintigraphy and similar radiographic techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of applicant's copending applicationSer. No. 850,299, filed Mar. 19, 1986, now abandoned, which is acontinuation-in-part of application Ser. No. 632,383, filed July 19,1984, now abandoned. Copending application Ser. No. 850,299 is the U.S.national phase of International PCT Application No. PCT/US85/01333,filed July 15, 1985.

FIELD OF THE INVENTION

The present invention relates to a dihydropyridine←→pyridinium salt typeof redox, or chemical, delivery system for the site-specific and/orsite-enhanced delivery of a radionuclide to the brain and other organs.More particularly, this invention relates to the discovery that achelating agent capable of chelating with a radionuclide and having aprimary, secondary or tertiary amino function can be converted to thecorresponding analogue in which said function is replaced with adihydropyridine←→pyridinium salt redox system and then complexed with aradionuclide to provide a new radiopharmaceutical that, in its lipoidaldihydropyridine form, penetrates the blood-brain barrier ("BBB") andallows increased levels of radionuclide concentration in the brain,particularly since oxidation of the dihydropyridine moiety in vivo tothe ionic pyridinium salt retards elimination from the brain whileelimination from the general circulation is accelerated.

The present radionuclide delivery system is well suited for use inscintigraphy and similar radiographic techniques.

BACKGROUND OF THE INVENTION

Radiographic techniques such as scintigraphy, and the like, findapplication in biological and medical procedures for diagnosis as wellas research. Scintigraphy involves the use of radiopharmaceuticals;i.e., compounds containing (or labeled with) a radioisotope (i.e.radionuclide) which upon introduction into a mammal become localized inspecific organs, tissue, or skeletal material that are sought to beimaged. When the radiopharmaceutical is so localized, traces, plates, orscintiphotos of the existing distribution of the radionuclide may bemade by various radiation -detectors known in the art. The observeddistribution of the localized radionuclide can then be used to detectthe presence of pathological conditions, abnormalities, and the like.Radiopharmaceuticals are thus often referred to as radiodiagnostics.

In many cases, radiopharmaceuticals are prepared using target-specificchelating agents which provide a bridge connecting a radionuclide, suchas a radioactive metal like technetium-99m, or the like, and a materialwhich will temporarily localize in the organ, tissue, or skeletalmaterial which is to be imaged. Typical chelating agents for suchpurposes are: polydentate ligands that form a 1:1 or 2:1 ligand:radioactive metal complex; macrocyclic ligands of appropriate ring sizeand preferably where all coordinating atoms are in a planarconfiguration; and bicyclic or polycyclic ligands that can encapsulatethe radioactive metal.

It is a well established fact that the delivery of drugs, includingradiopharmaceuticals, to the brain is often seriously limited bytransport and metabolism factors and, more specifically, by thefunctional barrier of the endothelial brain capillary wall deemed theblood-brain barrier or BBB. Site-specific delivery and/or sustaineddelivery of drugs to the brain are even more difficult.

A dihydropyridine←→pyridinium redox system has now been successfullyapplied to delivery to the brain of a number of drugs. Generallyspeaking, according to this system, a dihydropyridine derivative of abiologically active compound is synthesized, which derivative can enterthe CNS through the blood-brain barrier following its systemicadministration. Subsequent oxidation of the dihydropyridine species tothe corresponding pyridinium salt leads to delivery of the drug to thebrain.

Two main approaches have been used thus far for delivering drugs to thebrain using this redox system. The first approach involves derivation ofselected drugs which contain a pyridinium nucleus as an integralstructural component. This approach was first applied to delivering tothe brain N-methylpyridinium-2-carbaldoxime chloride (2-PAM), the activenucleus of which constitutes a quaternary pyridinium salt, by way of thedihydropyridine latentiated prodrug form thereof. Thus, a hydrophiliccompound (2-PAM) was made lipoidal (i.e. lipophilic) by making itsdihydropyridine form (Pro-2-PAM) to enable its penetration throughlipoidal barriers. This simple prodrug approach allowed the compound toget into the brain as well as other organs, but this manipulation didnot and could not result in any brain specificity. On the contrary, suchapproach was delimited to relatively small molecule quaternarypyridinium ring-containing drug species and did not provide the overallideal result of brain-specific, sustained release of the desired drug,with concomitant rapid elimination from the general circulation,enhanced drug efficacy and decreased toxicity. No "trapping" in thebrain of the 2-PAM formed in situ resulted, and obviously nobrain-specific, sustained delivery occurred as any consequence thereof:the 2-PAM was eliminated as fast from the brain as it was from thegeneral circulation and other organs. Compare U.S. Pat. Nos. 3,929,813and 3,962,447; Bodor et al, J. Pharm. Sci., 67, No. 5, pp. 685-687(1978); Bodor et al, Science, Vol. 190 (1975), pp. 155-156; Shek,Higuchi and Bodor, J. Med. Chem., Vol. 19 (1976), pp. 113-117. A morerecent extension of this approach is described by Brewster, DissertationAbstracts International, Vol. 43, No. 09, March 1983, p. 2910B. See alsoBodor et al, Science, Vol. 214, Dec. 18, 1981, pp. 1370-1372.

The second approach for delivering drugs to the brain using the redoxsystem involves the use of a pyridinium carrier chemically linked to abiologically active compound. Bodor et al, Science, Vol. 214, Dec. 18,1981, pp. 1370-1372, outlines a scheme for this specific and sustaineddelivery of drug species to the brain, as depicted in the followingScheme A: ##STR1## According to the scheme in Science, a drug [D] iscoupled to a quaternary carrier [QC]⁺ and the [D-QC]⁺ which results isthen reduced chemically to the lipoidal dihydro form [D-DHC]. Afteradministration of [D-DHC] in vivo, it is rapidly distributed throughoutthe body, including the brain. The dihydro form [D-DHC]

is then in situ oxidized (rate constant, k₁) (by the NAD←→NADH system)to the ideally inactive original [D-QC]⁺ quaternary salt which, becauseof its ionic, hydrophilic character, should be rapidly eliminated fromthe general circulation of the body, while the blood-brain barriershould prevent its elimination from the brain (k₃ >>k₂ ; k₃ >>k₇).Enzymatic cleavage of the [D-QC]⁺ that is "locked" in the brain effectsa sustained delivery of the drug species [D], followed by its normalelimination (k₅), metabolism. A properly selected carrier [QC]⁺ willalso be rapidly eliminated from the brain (k₆ >k₂). Because of thefacile elimination of [D-QC]⁺ from the general circulation, only minoramounts of drug are released in the body (k₃ >>k₄); [D] will be releasedprimarily in the brain (k₄ >k₂). The overall result ideally will be abrain-specific sustained release of the target drug species.Specifically, Bodor et al worked with phenylethylamine as the drugmodel. That compound was coupled to nicotinic acid, then quaternized togive compounds of the formula ##STR2## which were subsequently reducedby sodium dithionite to the corresponding compounds of the formula##STR3## Testing of the N-methyl derivative in vivo supported thecriteria set forth in Scheme A. Bodor et al speculated that varioustypes of drugs might possibly be delivered using the depicted oranalogous carrier systems and indicated that use of N-methylnicotinicacid esters and amides and their pyridine ring-substituted derivativeswas being studied for delivery of amino- or hydroxyl-containing drugs,including small peptides, to the brain. No other possible specificcarriers were disclosed. Other reports of this work with the redoxcarrier system have appeared in The Friday Evening Post, Aug. 14, 1981,Health Center Communciations, University of Florida, Gainesville, Fla.;Chemical & Engineering News, Dec. 21, 1981, pp. 24-25; and Science News,Jan. 2, 1982, Vol. 121, No. 1, page 7. More recently, the presentinventor has substantially extended the redox carrier system in terms ofpossible carriers and drugs to be delivered; see, for exampleInternational Patent Application No. PCT/US83/00725, filed by UNIVERSITYOF FLORIDA on May 12, 1983 and published under International PublicationNo. WO83/03968 on Nov. 4, 1983.

Nevertheless, serious need also has existed in this art for new,centrally acting drugs which can be site-specifically and sustainedlydelivered to the brain, while at the same time avoiding the aforesaidnoted and notable disadvantages and drawbacks associated withpenetration of the blood-brain barrier, with dihydropyridine latentiatedprodrug forms of drug species themselves comprising a pyridinium saltactive nucleus, with the necessity for introducing criticallycoordinated and designed, release rate-controlling substituents onto anyparticular drug carrier moiety, and/or with the limitation of deliveryof only known drug entities. This need has led to a new approach fordelivering drugs to the brain using the redox system. This novelapproach provides new derivatives of centrally acting amines in which aprimary, secondary or tertiary amino function has been replaced with adihydropyridine/pyridinum salt redox system. These new dihydropyridineanalogues are characterized by the structural formula ##STR4## wherein Dis the residue of a centrally acting primary, secondary or tertiaryamine, and ##STR5## is a radical of the formula ##STR6## wherein thedotted line in formula (i) indicates the presence of a double bond ineither the 4 or 5 position of the dihydropyridine ring; the dotted linein formula (ii) indicates the presence of a double bond in either the 2or 3 position of the dihydroquinoline ring system; m is zero or one; nis zero, one or two; p is zero, one or two, provided that when p is oneor two, each R in formula (ii) can be located on either of the two fusedrings; q is zero, one, or two, provided that when q is one or two, eachR in formula (iii) can be located on either of the two fused rings; andeach R is independently selected from the group consisting of halo, C₁-C₇ alkyl, C₁ -C₇ alkoxy, C₂ -C₈ alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C₁-C₇ haloalkyl, C₁ -C₇ alkylthio, C₁ -C₇ alkylsulfinyl, C₁ -C₇alkylsulfonyl, CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl, and --CONR'R"wherein R' and R", which can be the same or different, are each H or C₁-C₇ alkyl.

The new dihydropyridine analogues described in the preceding paragraphact as a delivery system for the corresponding quaternary compounds invivo; the quaternary derivatives, which also are chemical intermediatesto the dihydro compounds, are pharmacologically active and arecharacterized by site-specific and sustained delivery to the brain whenadministered via the corresponding dihydropyridine form. Nevertheless, aserious need still exists for an effective genera method for thesite-specific and/or sustained delivery of a desired radionuclide to thebrain. It would therefore be desirable to adapt the analogue concept tothe radiopharmaceutical area.

Summary of the Invention

It has now been found that a chemical delivery system based upon adihydropyridine←→pyridinium salt type redox system is uniquely wellsuited for an effective site-specific and/or sustained and/or enhanceddelivery of a radionuclide to the brain or like organ, via novel redoxsystem-containing radiopharmaceuticals, and novel redoxsystem-containing chelating agents and novel redox system-containingprecursors thereto, useful in the preparation of saidradiopharmaceuticals. In one aspect, the present invention thus providesnovel redox system-containing chelating agent precursors having theformula ##STR7## wherein ##STR8## is the residue of a chelating agentcapable of chelating with a metallic radionuclide, said chelating agenthaving at least one primary, secondary or tertiary amino functionalgroup, said functional group being not essential for the complexingproperties of said chelating agent, said residue being characterized bythe absence of at least one of said primary, secondary or tertiary aminofunctional groups from the chelating agent; y is 1 or 2; ##STR9## is aradical of the formula ##STR10## wherein n is zero, one or two; p iszero, one or two provided that when p is one or two, each R in formula(b) can be located on either of the two fused rings; q is zero, one, ortwo, provided that when q is one or two, each R in formula (c) can belocated on either of the two fused rings; and each R is independentlyselected from the group consisting of halo, C₁ -C₇ alkyl, C₁ -C₇ alkoxy,C₂ -C₈ alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C -C₇ haloalkyl, C₁ -C₇alkylthio, C₁ -C₇ alkylsulfinyl, C₁ -C₇ alkylsufonyl, --CH=NOR'" whereinR'" is H or C₁ -C₇ alkyl, and -CONR'R" wherein R' and R", which can bethe same or different, are each H or C₁ -C₇ alkyl; X⁻ is the anion of apharmaceutically acceptable organic or inorganic acid; t is the valenceof the acid anion; and s is a number which when multiplied by t is equalto y.

In another aspect, the present invention provides novel redoxsystem-containing chelating agents having the formula ##STR11## and thenon-toxic pharmaceutically acceptable salts thereof, wherein ##STR12##and y are defined as above, and ##STR13## is a radical of the formula##STR14## wherein the dotted line in formula (i) indicates the presenceof a double bond in either the 4 or 5 position of the dihydropyridinering; the dotted line in formula (ii) indicates the presence of a doublebond in either the 2 or 3 position of the dihydroquinoline ring system;m is zero or one; n is zero, one or two; p is zero, one or two, providedthat when p is one or two, each R in formula (ii) can be located oneither of the two fused rings; q is zero, one, or two, provided thatwhen q is one or two, each R in formula (iii) can be located on eitherof the two fused rings; and each R is independently selected from thegroup consisting of halo, C₁ -C₇ alkyl, C₁ -C₇ alkoxy, C₂ -C₈alkoxycarbonyl, C₂ -C₈ alkanoyloxy, C₁ -C₇ haloalkyl, C₁ -C₇ alkylthio,C₁ -C₇ alkylsulfinyl, C₁ -C₇ alkylsufonyl, --CH═NOR'" wherein R'"is H orC₁ -C₇ alkyl, and --CONR'R" wherein R' and R", which can be the same ordifferent, are each H or C₁ -C₇ alkyl.

In yet another aspect, the present invention provides, as an effectiveradionuclide delivery system, novel redox system-containingradiopharmaceuticals of the formula ##STR15## and the non-toxicpharmaceutically acceptable salts thereof, wherein M is a metallicradionuclide and the remaining structural variables are defined asbefore; in other words, (III) is the chelated, or complexed, counterpartof (II), formed by complexing the novel redox system-containingchelating agent of formula (II) with a radioactive metal. When aradiopharmaceutical of formula (III) is administered, due to itslipoidal nature it readily penetrates the BBB. Oxidation of (III) invivo affords the corresponding pyridinium salt of the formula ##STR16##wherein the structural variables are as defined above. Because of itshydrophilic, ionic nature, the formula (IV) substance is "locked-in" thebrain, thus allowing radiographic imaging of the radionuclide present inthe complex (IV). There is no readily biologically cleavable bondbetween the redox portion of the formula (IV) complex and theradiolabeled chelate portion thereof. Consequently, it is not expectedthat the quaternary "locked in" form will gradually cleave to releasethe redox moiety and the chelate portion of the molecule. Rather,sustained levels of the formula (IV) quaternary will be present at thedesired site.

It is generally considered most desirable, from the standpoint ofpatient and technician safety, to image the target area as soon aspossible after administration and to use relatively short-livedradioisotopes. Under these circumstances, or indeed even when longerlived radioisotopes are utilized, the "locked-in" quaternary form is notexpected to be metabolized or to exit the brain until after theradioactivity has decayed to a considerable extent. Thus, the presentinvention does not in fact provide a system for delivery and imaging ofpreviously known radiopharmaceuticals; by the time the present deliverysystem would no longer be in its "locked in" quaternary form, it wouldgenerally no longer be sufficiently radioactive for practical imaging.Thus, in contrast to the teachings of the Bodor et al publications. e.g.Bodor et a1. Science, Vol. 214, Dec. 18, 1981. pp. 1370-1372, whichemphasize the desirability of an inactive quaternary form locked in thebrain, the present invention provides, and indeed requires, an activequaternary form locked in the brain in order to allow effectiveradionuclide imaging.

Technetium-99m is a preferred radionuclide for diagnostic purposesbecause of its favorable radiation energy, its relatively shorthalf-life, and the absence of corpuscular radiation, and is preferredfor use in the present invention. Other radionuclides that can be useddiagnostically herein in a chelated form are cobalt-57, gallium-67,gallium-68, indium-111, indium-111m, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are applicable:

The term "drug" as used herein means any substance intended for use inthe diagnosis, cure, mitigation, treatment or prevention of disease inman or other animal.

The expression "non-toxic pharmaceutically acceptable salts" as usedherein generally includes the non-toxic salts of products of theinvention of structures (II) and (III) hereinabove formed withnon-toxic, pharmaceutically acceptable inorganic or organic acids of thegeneral formula HX. For example, the salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic, fumaric,methanesulfonic, toluenesulfonic and the like. The expression "anion ofa pharmaceutically acceptable organic or inorganic acid" as used herein,e.g. in connection with structures (I) and (IV) above, is intended toinclude anions of such HX acids.

The term "halo" encompasses fluoro, chloro, bromo and iodo.

The term "C₁ -C₇ alkyl" includes straight and branched lower alkylradicals having up to seven carbon atoms. When R, R', R" and/or R'" areC₁ -C₇ alkyl, they are preferably methyl or ethyl.

The term "C₁ -C₇ alkoxy" includes straight and branched chain loweralkoxy radicals having up to seven carbon atoms. When R is C₁ -C₇alkoxy, it is preferably methoxy or ethoxy.

The term "C₂ -C₈ alkoxycarbonyl" designates straight and branched chainradicals of the formula ##STR17## wherein the C₁ -C₇ alkyl group isdefined as above. When R is alkoxycarbonyl, it is preferablyethoxycarbonyl or isopropoxycarbonyl.

The term "C₂ -C₈ alkanoyloxy" designates straight and branched chainradicals of the formula ##STR18## wherein the C₁ -C₇ alkyl group isdefined as above. When R is alkanoyloxy, it is preferably acetoxy,pivalyloxy or isobutyryloxy.

The term "C₁ -C₇ haloalkyl" designates straight and branched chain loweralkyl radicals having up to seven carbon atoms and bearing one or morehalo substituents (F, Cl, Br or I), which can be the same or different.Preferably, when R is haloalkyl, the group contains 1 or 2 carbon atomsand bears 1 to 3 halogen substituents, e.g. chloromethyl ortrifluoromethyl.

The term "C₁ -C₇ alkylthio" includes straight and branched chainradicals of the type

    (C.sub.1 -C.sub.7 alkyl) --S--

wherein C₁ -C₇ alkyl is defined as before. When R is alkylthio, it ispreferably methylthio.

The terms "C₁ -C₇ alkylsulfinyl" and "C₁ -C₇ alkylsulfonyl" designateradicals of the formulas

    (C.sub.1 -C.sub.7 alkyl) --SO--

and

    (C.sub.1 -C.sub.7 alkyl) --SO.sub.2 --,

respectively, wherein C₁ -C₇ alkyl is defined as before. When R isalkylsulfinyl or alkylsulfonyl, methylsulfinyl and methylsulfonyl arepreferred.

When R is --CH═NOR'", it is preferably --CH═NOH or CH═NOCH₃. When R is--CONR'R", it is preferably --CONH₂ or --CON(CH₃)₂.

In formulas (I) through (IV) hereinabove, y is preferably l; n, m, p orq is preferably one; and R is preferably located in the 3-position instructures (a), (b), (i) or (ii) and in the 4-position in structures (c)or (iii). R is preferably --CH=NOR'" or --CONR'R" wherein R', R" and R'"are as broadly defined hereinabove. Most preferably, R is --CONH₂ orCH═NOCH₃.

The expression "residue of a chelating agent capable of chelating with ametallic radionuclide, said chelating agent having at least one primary,secondary or tertiary amino functional group, said functional groupbeing not essential for the complexing properties of said chelatingagent, said residue being characterized by the absence of at least oneof said primary, secondary or tertiary amino functional groups from thechelating agent" is believed to be self-explanatory. By way of example,if a chelating agent having a primary amino function which isnon-essential in terms of chelating ability can be represented by thestructural formula ##STR19## then the corresponding residue could bedepicted as ##STR20## in formulas (I) through (IV), the ring nitrogenatoms in structures (a) through (c) and (i) through (iv) are thuslocated in the same position as was the nitrogen atom of the originalamino function. As a specific example, in the case of a chelating agenthave the structure ##STR21## the corresponding residue would be##STR22## and the corresponding redox system-containing chelating agentprecursor of formula (I) would have the structure ##STR23## wherein y=1and ##STR24## s, X⁻ and t are defined as with formula (I). Similarly,when the chelating agent has the structure ##STR25## then thecorresponding residue is ##STR26## and the corresponding redoxsystem-containing chelating agent precursor of formula (I) would havethe structure ##STR27## wherein y=1 and the other structural variablesare defined as with formula (I).

As another example, when the chelating agent has the structure ##STR28##wherein R¹, R², R³ and R⁴ are each H or C₁ -C₃ alkyl and n' is aninteger of 0 to 3, then the corresponding residue is ##STR29##respectively; and the corresponding redox system-containing chelatingagent precursor of formula (I) would have the structure ##STR30##respectively, wherein y=1 and s, X⁻ and t are as defined with formula(I) and R¹, R² and n' are as defined immediately above.

It will be apparent from the foregoing that the exact structure of theamino function in the chelating agents is immaterial insofar as concernsthe structure of the instant derivatives of formulas (I) through (IV),for in formulas (I) through (IV) the entire amino function in the parentchelating agents has been replaced with a dihydropyridine/pyridiniumsalt redox system. Thus, virtually any chelating agent capable ofcomplexing with a radionuclide and having at least one primary,secondary or tertiary amine functional group which is non-essential interms of chelating properties can provide the chelating agents residue##STR31## in the instant derivatives. Many illustrative such aminegroups will be apparent to those skilled in the art; most commonly,however, the chelating agent's functional group which is to be replacedwith the redox system is simply an --NH₂ group. And such amino group canbe readily introduced into the structure of a known chelating agent notalready comprising same and then replaced by the instant redox system togive the desired derivatives, as described in more detail hereinbelow.

It too will be appreciated that the radical represented by ##STR32## informulas (II) and (III) must enable the complex of formula (III) topenetrate the BBB and must also be capable of being oxidized in vivo tothe corresponding quaternary structure. The ionic entity which resultsfrom such in vivo oxidation is prevented from efflux from the brain,while elimination from the general circulation is accelerated. Incontradistinction to the drug-carrier entities disclosed, for example,in Science, Vol. 214, Dec. 18, 1981, pp. 1370-1372, however, there is noreadily metabolically cleavable bond between drug and quaternaryportions; the active species delivered in the present case is theformula (IV) quaternary itself.

It will also be appreciated that a compound of formula (III) may beadministered as the free base or in the form of a non-toxicpharmaceutically acceptable salt thereof, i.e. a salt which can berepresented by the formula ##STR33## wherein M, ##STR34## y and HX aredefined as before; and that, regardless of the actual form in which thecompound is administered, it will be converted in vivo to a quaternarysalt of formula (IV), the anion X⁻ being present in vivo. It is notnecessary that the anion be introduced as part of the compoundadministered. Indeed, even when the compound of formula (III) is used inits salt form, the anion of the formula (IV) compound is not necessarilythe same as that present in the formula (III) compound. Indeed, theexact identity of the anionic portion of the compound of formula (IV) isimmaterial to the in vivo transformation of (III) to (IV).

Insofar as concerns the expression "said functional group being notessential for the complexing properties of said chelating agent", itwill be apparent that this expression is intended to mean that anyprimary, secondary or tertiary amino functional group in a chelatingagent which can be replaced with the instant redox system withoutdestroying the chelating agent's ability to complex with theradionuclide is considered herein to be not essential for complexingproperties. On the other hand, replacement of an amino functional groupwhich would lead to a redox system-containing structure which would beincapable of complexing with a radionuclide is not within the ambit ofthis invention.

In accord with the present invention, the sustained delivery of aradionuclide to the brain in sufficient concentrations for radioimagingcan be effected with much lower concentrations in the peripheralcirculation and other tissues. The present invention of course willallow such imaging of any other organs or glands in which sufficientradioactivity accumulates. Thus, for example, it is expected that thequaternary form (IV) which is locked in the brain will be locked in thetestes as well.

The novel radionuclide delivery system of this invention begins with thepreparation of the novel redox system-containing chelating agentprecursors of formula (I). The preparation of those precursors will betailored to the particular chelating portion and redox portion to becombined, as well as to the presence or absence of other reactivefunctional groups (amino, mercapto, carboxyl, hydroxy) in either thechelating or redox portion. Typically, if such other reactive groups arepresent, they are found in the chelating portion. In any event, whensuch groups are present and it is desired to protect them, a step thatintroduces appropriate protecting groups can be incorporated at asuitable stage of the synthetic pathway. Protective groups are wellknown in the art and include t-butoxycarbonyl for amino groups,N-methyleneacetamido for mercaptans, and N-hydroxysuccinimidyl forcarboxyl groups. Acyl or carbonate groups are typically utilized toprotect alcohol hydroxyls. When carbonate protecting groups are desired,the step of introducing the protecting groups will involve reacting thealcohol with a halocarbonate of the type ROCOCl or ROCOBr (formed byreaction of ROH with COCl₂ or COBR₂), R typically being lower alkyl. Foracyl protecting groups, the alcoholic hydroxyl is reacted with an acylhalide R'Cl or R'Br, R' being --COCH₃ or --COC(CH₃)₃. Yet other reactionschemes and reactants will be readily apparent to those skilled in theart as will the appropriate means for removing such protective groupsafter they have achieved their function and are no longer needed.

In forming the precursors of formula (I), at least one primary,secondary or tertiary amino functional group in a chelating agent willbe replaced with ##STR35## the hydrophilic, ionic pyridinium salt formof the dihydropyridine←→pyridinium salt redox system.

It will be appreciated that by ##STR36## there is intended any non-toxicredox moiety of structure (a), (b) or (c) hereinabove comprising,containing or including the pyridinium nucleus, whether or not a part ofany larger basic nucleus, and whether substituted or unsubstituted, theonly criterion therefor being capacity for chemical to the correspondingdihydropyridine form ##STR37## BBB-penetration of ##STR38## and in vivooxidation of ##STR39## back to the quaternary pyridinium salt redoxmoiety

As aforesaid, the ionic pyridinium salt radiopharmaceutical/redox entityof formula (IV) which results from in vivo oxidation of thedihydropyridine form (III) is prevented from efflux from the brain,while elimination from the general circulation is accelerated.Radioimaging of the radionuclide present in the "locked in" formula (lV)quaternary allows observation of the distribution of the localizedradionuclide for diagnosis of pathological conditions, abnormalities,etc.

The following synthetic schemes illustrate various approaches to thepreparation of the redox system-containing chelating agent precursors offormula (1), to the corresponding redox system-containing chelatingagents of formula (II) and to the corresponding redox system-containingradiopharmaceuticals of formula (III). Also shown are the corresponding"locked in" quaternaries of formula (IV) formed by in vivo oxidation ofthe formula (III) chelates, said formula (IV) quaternaries being theprimary localized materials whose radionuclide content is imaged byradiation detection means. ##STR40##

Thus, Schemes 1 (version 1), 5 ) versions 1 and 2) and 9 (version 1)above illustrate typical conversion of a carboxylic acid ester group tothe corresponding amide (--CONH₂); reduction of the amide function tothe corresponding amine (--CH₂ NH₂); replacement group with the desiredquaternary function ##STR41## utilizing a Zincke reagent; and reductionresultant quaternary of formula (I) to the corresponding dihydro offormula (II), or conversion of (I) directly to the formula (III)radiopharmaceutical. Variations of this type of reaction sequence areshown in versions 2 and 3 of Scheme 1, version 3 of Scheme 5 and version2 of Scheme 9, in which a protecting group is introduced prior toreaction with the Zincke reagent and then removed prior to reduction ofthe quaternary function. In the case of the chelating agents shown inthese schemes, reaction with acetone protects both the secondary aminoand thiol functions by formation of thiazolidine structures so thatthose functions do not interfere in the reaction with the Zinckereagent. Subsequently, the secondary amino and mercapto groups areregenerated by reacting the protected intermediate with mercuricchloride in an organic solvent such as methanol, conveniently at roomtemperature, and then decomposing the resulting complex with hydrogensulfide. See, for example British Patent Specification No. 585,250,which utilizes such a procedure for the production of esters ofpenicillamine.

Schemes 2 (version 1), 4 and 10 above illustrate typical conversion ofan alcohol (--CH₂ OH). which may be obtained from the correspondingcarboxylic acid ester, to the corresponding halide (--CH₂ Cl or --CH₂Br); reaction of the halo derivative with the appropriate pyridinederivative ##STR42## to afford the desired formula (I) quaternary; andreduction to the corresponding formula (II) dihydro or conversiondirectly to the corresponding formula (III) radiopharmaceutical. Schemes4 and 10 also illustrate removal of a protecting group immediately afterformation of the quaternary while version 2 of Scheme 2 illustratesintroduction and removal of the thiazolidine protecting group discussedabove with respect to versions 2 and 3 of Scheme 1, etc.

In Scheme 3 above, there is shown a typical method for introducting alonger alkylene chain between an atom which is involved in forming thechelate structure and a pendant NH₂ group which is to be replaced withthe quaternary structure. As depicted in this scheme a secondary aminogroup >NH is reacted with a haloalkamide, e.g. BrCH₂ CONH₂, replacingthe hydrogen of the >NH with --CH₂ CONH₂. Reduction of the amide affordsthe corresponding >NCH₂ CH₂ NH₂ compound. That amine can then be reactedwith a Zincke reagent to replace the --NH₂ with ##STR43## followed byreduction as in the other schemes; preferably, however, any free thiolgroups are protected prior to reaction with the Zincke reagent.

Schemes 6, 11 and 12 illustrate yet other methods for lengthening thealkylene chain, the chain here being interrupted by one or more oxygenatoms. Thus, a --CH₂ OH group is typically converted to thecorresponding lithium salt and then reacted with an iodoalkanol, e.g.ICH₂ CH₂ OH, to convert the --CH₂ O--Li⁺ group to a --CH₂ OCH₂ CH₂ OHgroup. [Obviously, the chain could be lengthened by utilizing alonger-chain iodoalkanol, or by repeating the two steps just described(in which case additional intervening oxygen atoms would beintroduced.)] The --CH₂ OCH₂ CH₂ OH group is then converted to thecorresponding --CH₂ OCH₂ CH₂ Br or --CH₂ OCH₂ CH₂ Cl, which is thenreacted with the selected pyridine derivative ##STR44## to form thedesired quaternary salt. In the schemes shown, a protecting group isremoved immediately after quaternization to afford the formula (I)quaternary, which is subsequently reduced as in the other schemes.

In Schemes 7, 8 and 13 above, replacement of an --NH₂ group with thecorresponding quaternary ##STR45## is shown, utilizing a Zincke reagent.Where appropriate, quaternary formation is followed by removal ofprotecting groups, as in Schemes 7 and 13. The resultant formula (1)quaternary is then reduced as shown in the other schemes.

Many of the earliest steps in the reaction schemes depicted aboveparallel reactions described in Fritzberg U.S. Pat. No. 4,444,690. See,for example, the conversion of 14 to 15 in Scheme 2; the conversion of16 to 32 to 33 in Scheme 4; the conversion of 15 to 40 to 41 in Scheme5; the conversion of 56 to 57 to 58 and the conversion of 60 to 61 inScheme 6; and so on.

Similar schemes can be shown for the preparation of the otherderivatives of this invention. The steps of introducing and removingprotecting groups are only included when necessary. Also, the order ofsteps may be altered; in particular, quaternization may occur earlier inthe reaction scheme, depending of course on the particular compoundsinvolved. Other reaction schemes, reactants, solvents, reactionconditions, etc. will be readily apparent to those skilled in the art.Also, insofar as concerns the quaternary derivatives, when an aniondifferent from that obtained is desired, the anion in the quaternarysalt may be subjected to anion exchange via an anion exchange resin or,more conveniently, by use of the method of Kaminski et al, Tetrahedron,Vol. 34, pp. 2857-2859 (1978). According to the Kaminski et al method, amethanolic solution of an HX acid will react with a quaternary ammoniumhalide to produce the methyl halide and the corresponding quaternary Xsalt.

The processes exemplified by Schemes 1, 3, 5, 7, 8, 9 and 13 include thestep of reacting a compound containing an --NH₂ group with a Zinckereagent. The Zincke reaction can be to derive the instant derivatives inwhich ##STR46## is the residue of a primary amine, or their protectedcounterparts, directly from the corresponding primary amine/parentchelating agent. However, if it is des prepare the instant derivativesin which ##STR47## is the residue of a secondary or tertiary amine, orprotected counterparts, via the Zincke reaction, then one will not usethe parent secondary or tertiary amine chelating agent as the startingmaterial but would instead use the corresponding primary amine as thestarting material. Alternatively, a compound of the formula ##STR48##wherein Hal is chloro or bromo and ##STR49## is the residue of thechelating agent as defined or its protected counterpart can be reactedwith a pyridine derivative of the formula ##STR50## wherein R, n, p andq are defined with formula (I), e.g. nicotinamide, isonicotinamide,picolinamide, 3-quinolinecarboxamide, 4-isoquinolinecarboxamide or thecorresponding oximes in which a --CH═NOCH₃ group is present in place ofthe --CONH₂ group of nicotinamide etc. See, for example, Schemes 2, 4,6, 10, 11 and 12. The starting pyridine derivatives are readilyavailable or can be prepared in known manner, e.g.3-quinolinecarboxamide can be prepared by treating the correspondingacid with ammonia.

When a Zincke reagent is utilized in the reaction sequence, such reagentcan be prepared by reacting 1-chloro-2,4-dinitrobenzene with a compoundof the formula ##STR51## wherein R, n, p and q are defined as withformula (I), to afford the corresponding Zincke reagent of the formula##STR52## respectively. Thus, for example, the specific Zincke reagentdepicted in Scheme 7 can be prepared by reacting nicotinamide with1-chloro-2,4-dinitrobenzene. See also Zincke et al, Annalen, 1904, 333,296; Lettre et al, Annalen, 1953, 579, 123; Keijzer et al, Heterocycles,Vol. 16, No. 10, 1981, 1687. Preferred Zincke reagents are those inwhich n, p or q is one and R is --CONH₂ or --CH=NOCH₃ and is located inthe 3-position of the pyridinium or quinolinium structure or in the4-position of the isoquinolinium structure. Typically, the Zinckereagent is reacted with the primary amine, which may be veryconveniently employed in the form of its acid addition salt, in thepresence of a suitable base, e.g. triethylamine, in an appropriateorganic solvent, e.g. methanol, to afford the desired quaternary salt.

When a starting material of the formula ##STR53## wherein ##STR54## andHal are defined above is utilized to prepare the quaternary salt, saidstarting material can be prepared from the corresponding alcohol, e.g.by methods such as those depicted in Schemes 2, 4, 6, 10, 11 or 12.

Reduction of the quaternary salt of formula (I) to the correspondingdihydro derivative of formula (II) can be conducted at a temperaturefrom about -10° C. to room temperature, for a period of time from about10 minutes to 2 hours, conveniently at atmospheric pressure. Typically,a large excess of reducing agent is employed. e.g., a 1.5 molar ratio ofreducing agent to starting compound of formula (I). The process isconducted in the presence of a suitable reducing agent, preferably analkali metal dithionite such as sodium dithionite or an alkali metalborohydride such as sodium borohydride or lithium aluminum borohydride,in a suitable solvent. Sodium dithionite reduction is convenientlycarried out in an aqueous solution; the dihydro product of formula (II)is usually insoluble in water and thus can be readily separated from thereaction medium. In the case of sodium borohydride reduction, an organicreaction medium is employed, e.g., a lower alkanol such as methanol, anaqueous alkanol or other protic solvent. More typically, however, thequaternary of formula (I) is reduced in the same reaction mixture as thereduction of the radionuclide (preferably technetium) to an appropriateoxidation state, affording the formula (III) radiopharmaceutical in onestep from the formula (I) quaternary. Further details of the one-stepreduction are given hereinbelow.

It will be apparent from the foregoing that a wide variety ofderivatives of formulas (I) through (IV) can be obtained in accord withthis invention. In a particularly preferred embodiment of thisinvention, however, there are provided novel chelating agent precursorsof the formula ##STR55## wherein each R₁ is independently selected fromthe group consisting of H and C₁ -C₇ alkyl, or an R₁ can be combinedwith the adjacent >C--R₁ such that ##STR56## represents >C═0; each R₂ isindependently selected from the group consisting of H and C₁ -C₇ alkyl,or an R₂ can be combined with the adjacent >C--R₂ such that ##STR57##represents >C═0; ##STR58## is a radical of the formula ##STR59## whereineach R₃ is independently selected from the group consisting of H and C₁-C₇ alkyl, alk is a straight or branched lower alkylene group ) whichadditionally may contain 1, 2 or 3 nonadjacent oxygen atoms in thechain, and ##STR60## is as defined with formula (I) hereinabove; X⁻ andt are as defined with formula (I); and s' is a number which whenmultiplied by t is equal to one. Preferably, the salts of formula (Ia)have the partial structure ##STR61## or are position isomers and/orhomologs of the first two partial structures shown. It is also preferredthat when ##STR62## then each R₃ is preferably H and alk is preferably aC₁ -C₆ alkylene group, or a C₁ -C₆ alkylene group interrupted by anoxygen atom in the chain; and that when ##STR63## then alk is preferablya C₁ -C₆ alkylene group, or a C₁ -C₆ alkylene group interrupted by anoxygen atom in the chain. Preferred values for ##STR64## in formula (Ia)are as given in conjunction with formula (I) hereinabove.

Corresponding to the preferred novel chelating agent precursors offormula (Ia) are the preferred novel chelating agents of the formula##STR65## wherein R₁ and R₂ are as defined with formula (Ia) and##STR66## is a radical of the formula ##STR67## wherein R₃ and alk areas defined with formula (Ia) and ##STR68## is as defined with formula(II) hereinabove. Preferred compounds of formula (IIa) are the dihydroderivatives corresponding to the preferred compounds of formula (IIa).

Likewise preferred are the novel radiopharmaceuticals in which a formula(IIa) compound is chelated with a radioactive metal, especially withtechnetium. Especially preferred radiopharmaceuticals have the formula##STR69## wherein R₁ and R₂ are as defined with formula (Ia) and##STR70## is a radical of the formula ##STR71## wherein R₃ and alk areas defined with formula (Ia) and ##STR72## is as defined with formula(II) hereinabove; and the corresponding quaternaries, especially thoseof technetium, "locked in" the brain, which have the formula ##STR73##wherein R₁, R₂, s', X⁻ and t are as defined with formula (Ia) and##STR74## is a radical of the formula ##STR75## wherein R₃ and alk areas defined with formula (Ia) and ##STR76## is as defined with formula(I) hereinabove. The preferred complexes of formulas (IIIa) and (IVa)are those which correspond to the preferred derivatives of formulas (Ia)and (IIa).

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given, it being understoodthat same are intended only as illustrative and in no wise limitative.

EXAMPLE 1

To a stirred solution containing 115.6 g (1.6 mol) of isobutyraldehyde 1in 184 g of carbon tetrachloride are added dropwise, at 40°-50° C., 108g (0.8 mol) of 97% sulfur monochloride. The addition is carried outduring a 2.5 hour period, under a nitrogen atmosphere, with occasionalcooling. The solution is maintained at 30°-45° C., with stirring, for anadditional 48 hour period, under a current of nitrogen, to remove thehydrogen chloride liberated. The solution is distilled under vacuum togive 72 g of the desired 3,4-dithia-2,2,5,5-tetramethylhexane-1,6-dione,i.e. Compound 2 of Scheme 1. ¹ H NMR(CDCl₃) δ 9.1(s,2--CHO),1.4[s,12,--C(CH₃)₂ --].

EXAMPLE 2

To 10 g (0.07 mol) of ethyl cyanoglyoxylate-2-oxime 3 are added 125 mLof absolute ethanol, 15 g of hydrogen chloride gas and 1 g of platinumoxide. The mixture is hydrogenated using a Parr-hydrogenation apparatus.Hydrogen uptake is complete in 3 hours. The product is removed byfiltration and taken up in 75 mL of hot 95% ethanol. The ethanolsolution is filtered. The filtrate is then cooled and the crystallineproduct which separates on standing is removed by filtration. There isthus obtained ethyl 2,3-(diammonium)propionate dichloride, i.e. Compound4 of Scheme 1. Yield 5 g (35%). melting point 164°-166° C. (lit.164.5°-65° C.): ¹ H NMR(D₂ O) δ 4.5(m.3.--NCHCO--, --OCH₂ CH₃),3.5(m,2,--NCH₂ CH--), 1.3(t,3,--OCH₂ CH₃).

EXAMPLE 3 Procedure I

To 1.0 g (5 mmol) of the bisaldehyde 2 is added dropwise a solution of1.0 g 15 mmol) of the ester 4 and 0.9 mL of pyridine in 30 mL ofmethanol at 0° C. while under a nitrogen atmosphere. The addition takesplace during a 10 minute period. The solution is then allowed to standfor 1 hour, after which time 10 mL of water is added. The solution turnsturbid and warms to 26° C. The solution is stirred for an additional 20minute period, after which time the white precipitate which formssettles out of solution. The precipitate is removed by filtration andthen is taken up in chloroform. The chloroform solution is dried oversodium sulfate. Removal of the solvent and trituration of the residuewith petroleum ether gives white plate-like crystals of the desiredproduct,5,8-diaza-1,2-dithia-6-ethoxycarbonyl-3,3,10,10-tetra-methylcyclodeca-4,8-diene,i.e. Compound 5 of Scheme 1, in 53% yield (1 g), melting point 98°-99°C. IR (thin film) 3450, 1740, 1650 cm⁻¹ ; ¹ H NMR(CDCl₃) δ6.9(m,2,c--N═CH--), 3.0-4.6(m.5.--OCH₂ CH₃, --NCH₂ CH--N--), 1.5[m,15,2>C(CH₃)₂, --OCH₂ CH₃ ].

Procedure II

To 1.0 g (5 mmol) of the bisaldehyde 2 in 10 mL of methanol is addeddropwise 1.0 g (5 mmol) of the ester 4 and 1 g (12 mmol) of sodiumbicarbonate in 20 mL of a 50:50 by volume mixture of methanol and water.The mixture is stirred at 0° C. for 10 minutes, after which time 10 mLof water is added. The resultant mixture is maintained at roomtemperature,. with stirring, for 2 hours. Water is added until the whiteprecipitate which forms separates out of solution. The precipitate isremoved by filtration and taken up in chloroform. Removal of the solventby rotary evaporation affords 0.4 g (21% yield) of Compound 5, having amelting point and ¹ H NMR spectrum identical to the product of ProcedureI.

Procedure III

A solution of 8 g of the ester 4 and 7 mL of pyridine in 200 mL ofmethanol is added dropwise over a two hour period to a solution of 8 gof bisaldehyde 2 in 25 mL of methanol. The reaction mixture is cooled inan ice bath after the addition for 1 hour, then is allowed to remain atroom temperature for 1 hour. The reaction mixture is then placed in afreezer (-20° C.) overnight. The solution is concentrated to one-thirdvolume, water is added and the aqueous solution is extracted withcloroform. The chloroform extract is washed with saturated aqueoussodium chloride solution and dried over magnesium sulfate. Removal ofthe solvent leaves a viscous mass, which is dissolved in 20 mL ofhexane. The hexane solution is cooled in an acetone/dry ice bath until awhite powder separates. The product is removed by filtration and takenup in chloroform. The chloroform solution is concentrated. Whitecrystals of Compound 5 are formed on standing. Yield 7 g. melting point95°-96° C. NMR and IR as in Procedure I.

EXAMPLE 4 Procedure I

A solution of 5 g of the ester 5 in 20 mL of tetrahydrofuran and 20 mLof aqueous ammonia is stirred at room temperature for 2 hours, afterwhich time it is allowed to stand at room temperature for 24 hours.Removal of solvent leaves a white powder which is removed by filtration.The product,6-carbamoyl-5.8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene,i.e. Compound 6- of Scheme 1, is crystallized from a mixture ofisopropanol and water. Yield 4 g (88%), melting point 181°-83° C. IR(KBr) 3300, 3100 1650 cm ⁻¹ ; ¹ H NMR (CDCl₃) δ 7.0(m,2, --HC═N--),6.4(broad band, 2, --CONH₂), 3.8-4.6[m,3,--NCH₂ --CH(N--)CO--], 1.5, 14[s, 12, >C(CH₃)₂)].

Procedure II

A solution of 5 g of the ester 5 in 20 mL of tetrahydrofuran, 20 mL ofethanol and 20 mL of aqueous ammonia (28%) was stirred at roomtemperature for 16 hours. Removal of the solvent leaves Compound 6 as awhite powder, which crystallizes from toluene as white plates. Yield 4g, melting point 193°-194° C. IR and NMR as in Procedure I.

EXAMPLE 5

To 3.7 g of the amide 6 in 25 mL of 95% ethanol is added 2 g of sodiumborohydride. The mixture is stirred at room temperature for 2 hours,then is heated at reflux for 2 hours. The solution is thereafterconcentrated in vacuo and water is added to precipitate the product. Thewhite crystalline product is removed by filtration. Recrystallizationfrom, a mixture of isopropanol and water affords6-carbamoyl-5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane, i.e.Compound 7 of Scheme 1, as fine white needles melting at 138°-139° C.Yield 3 g. 1H NMR(CDCL₃) δ 2.3-4.0[m,7, --NCH₂ CH--N--, 2--NCH₂--C(CH₃)--S--], 1.8(broad band, 2, --CONH₂),1.3[m,14, C(CH₃)₂),--CNH--CH₂ --].

EXAMPLE 6

A solution of 1.8 g of the amide 7 in 50 mL of dry tetrahydrofuran isadded dropwise to a slurry of 1 g of lithium aluminum hydride in 100 mLof dry tetrahydrofuran. The addition takes place over a 30 minuteperiod. The mixture is then heated at the reflux temperature for 20hours. At the end of that time, the reaction mixture is first cooled andthen quenched with saturated Na-K tartrate solution. The aqueous phaseis extracted with chloroform. The combined organic phase is then driedover sodium sulfate. Removal of the solvent by rotary evaporationaffords, as a viscous oil,5-aminomethyl-4,7-diaza-2,9-dimethyldecane-2,9-dithiol, i.e. Compound 8of Scheme 1; ¹ H NMR (CDCl3) δ 2.8[m,9,--NCH₂ CH--C(CH₂)NH--³¹, 2--NCH₂--C(CH(hd 3)₂ S--], 1.5[m,14,, >C(CH₃)₂, --SH].

EXAMPLE 7

Methoxyamine hydrochloride (5 g; 0.06 mol) is dissolved in 50 mL ofmethanol and the solution is neutralized to pH 6 with 1 M methanolicKOH. The resultant mixture is filtered and to the filtered solution isadded 3.2 g (0.03 mol) of 3-pyridinecarboxaldehyde. That mixture isheated at reflux for 4 hours. The methanol is evaporated and the solidis crystallized from a mixture of ethanol and water. There is thusobtained 0-methyl-3-pyridinealdoxime having the structural formula##STR77##

A mixture of 2.7 g (22 mmol) of 0-methyl-3-pyridinealdoxime and 7 g (33mmol) of 1-chloro-2,4-dinitrobenzene is maintained on a water bath for 1hour while being stirred to a red homogenous mixture. The resultantmixture is dissolved in 35 mL of methanol, treated with charcoal andfiltered. The filtrate is treated successively with two 100 mL portionsof ether. The ether mixture is stirred and the product is removed byfiltration. Obtained in this manner is3-[(methoxyimino)methyl]-1-(2,4-dinitrophenyl)pyridinium chloride, i.e.the Zincke reagent identified as Compound 9 in Schemes 1, 3, 5, 8, 9 and13.

EXAMPLE 8

A solution of 1.6 g (5 mmol) of the Zincke reagent 9 in 2 mL of methanolis added dropwise to 2.65 g (10 mmol) of the amine 8 in 2 mL ofmethanol. The reaction mixture is heated at reflux for 2 hours, afterwhich time ether is added to precipitate the product. Alternatively,amine 8 may be utilized as its hydrobromide salt and the reaction may beconducted in the presence of triethylamine (5-10 mmol). There is thusobtained1-{{2',3'-bis-{N-[(2"-mercapto-2"-methyl)propyl]amino}propyl}}-3-[(methoxyimino)-methyl]pyridiniumchloride, i.e. Compound 10 of Scheme 1.

EXAMPLE 9

To 3.15 g of the dialdehyde 2 is added 4.0 g of ethylenediamine, withstirring and cooling, over a period of 10 minutes. The thick mass whichresults is stirred for an additional one minute period, then allowed tostand for 1 hour at room temperature and subsequently cooled for 16hours in a freezer (-20° C.). The solid is removed by filtration andwashed with 500 mL of water. The white product is then taken up inchloroform and the chloroform solution is dried over sodium sulfate.Removal of the chloroform gives 2.5 g of5.8-diaza-1.2-dithia-3,3,10,10-tetramethylcyclodeca-4,8-diene, i.e.Compound 24 of Scheme 3, as a white crystalline product, melting at168°-170° C. (lit. 162°-164° C., 163°-166° C.). ¹ H NMR(CDCl₃) δ6.9(s,2,--HC═N--), 4.2,3.0(doublet of doublet. 2, 2--CH₂ --CH₂), 1.40[s,6,--C(CH₃)₂ --]Anal. Calcd. for C₁₀ C₁₈ N₂ S₂ : C, 52.13; H, 7.88; N,12.16; S, 27.83. Found: C, 52.20; H, 7.90; N, 12.14; S, 27.74.

EXAMPLE 10

A solution of 0.5 g of 24 and 0.3 g of sodium borohydride in 23 mL ofethanol is stirred- at room temperature for 1 hour, then is heated atthe reflux temperature for 20 minutes. Then, 10 mL of water are addedand the mixture is heated for an additional 10 minutes. The solvent ispartially removed by rotary evaporation and the residue is extractedthree times with 10 mL portions of chloroform. The chloroform extract isdried over sodium sulfate and the solvent is removed by rotaryevaporation. The resultant liquid solidifies on cooling. Flashchromatography (eluent hexanes/dichloromethane/isopropanol 5:1:1 byvolume) gives 5,8-diaza-1,2-dithia-3,3,10,10-tetramethylcyclodecane,i.e. Compound 25 of Scheme 3, as a solid, melting at 52°-53° C. ¹ HNMR(CDCl₃) δ 3-2.1(m,10 ring protons), 1.1,1.2(s,6 CH₃, CH₃).

EXAMPLE 11

N-(t-butoxycarbony1), N-(2-mercaptoethyl)glycyl homocysteine thiolactone67 is prepared as described in Examples 1 and 2 of Byrne et al U.S. Pat.No. 4,434,151, and is dissolved (1.0 gram; 3 millimoles) in 25milliliters of tetrahydrofuran (THF). The resulting solution is thencooled to about 0° C. and ethylenediamine (1.8 grams; 30 millimoles) isadded to form a new solution. The resulting new solution is maintainedfor about one hour. The volatile components of the solution arethereafter removed with a rotary evaporator. n-Butanol (about 10milliliters) is added to the "dried" solution components and the liquidcomponents of the resulting composition are again removed by rotaryevaporation. The last step is repeated until the vapors remaining in theevaporation vessel do not cause a moistened pH-indicator paper toindicate a basic pH value, thereby also indicating that theethylendiamine has been substantially removed and that theN-(t-butoxycarbonyl), N-(2-mercaptoethyl)-glycylN'-(2-aminoethyl)homocysteinamide, i.e. Compound 68 of Scheme 7, soobtained is substantially pure.

EXAMPLE 12

A mixture of 8 g (66 mmol) of nicotinamide and 20 g (99 mmol) of1-chloro-2,4-dinitrobenzene is maintained on a water bath for one hour,with stirring. The red homogenous mixture which results is dissolved in100 mL of methanol and decolorized with charcoal. The filtrate is thentreated with 100 mL of ether and the yellow product which separates isremoved by filtration and washed with 500 mL of ether. The highlyhygroscopic product, 1-(2,4-dinitrophenyl)-3-carbamoylpyridiniumchloride, is the Zincke reagent 69, employed for example in Scheme 7 andversion 3 of Scheme 1. ¹ H NMR (D₂ O) δ 8.5-10.0(m,7,ArH,Py-H).

EXAMPLE 13

The procedure of ExamPle 8 is substantially repeated, except that anequivalent quantity of N-(t-butoxycarbonyl), N-(2-mercaptoethyl)glycylN'-(2-aminoethyl)homocysteine (68) is used in place of the amine 8 andan equivalent quantity of 1-(2,4-dinitrophenyl)-3-carbamoylpyridiniumchloride (69) is used in place of the Zincke reagent 9. Obtained in thismanner is Compound 70 of Scheme 7.

EXAMPLE 14

Compound 70 (0.002 mol) is dissolved with stirring in absolute ethanol(50 milliliters) and cooled to about 0° C. in an ice-water bath. HCl gasis bubbled through the stirred solution for 15 minutes, and the solutionis thereafter stirred for an additional 15 minutes. Diethyl ether (200milliliters).is thereafter added to the solution to precipitate thesalt. The precipitate is filtered and washed with diethyl ether and thesolid is then dried in vacuo to provide the corresponding de-protectedquaternary, Compound 71 of Scheme 7.

EXAMPLE 15

N-[2-(S-acetamidomethyl)mercaptopropionyl)glycyl homocysteinethiolactone (Compound 77 of Scheme 8). prepared as described in Examples7 and 9 of Byrne et al U.S. Pat. No. 4,434,151, is suspended (1.0 gram;3 millimoles) in 25 milliliters of THF. The resulting suspension iscooled to a temperature of about 0° C. in an ice-water bath, andethylendiamine (1.8 grams; 30 millimoles) is added to form a newsolution. N-[2-(acetamidomethyl)mercaptopropionyl]-glycylN'-(2-aminoethyl)homocysteinamide, i.e. Compound 78 of Scheme 8, isthereafter obtained in a manner substantially similar to that describedin Example 11 for the analogous compound.

EXAMPLE 16

The procedure of Example 8 is substantially repeated, except that anequivalent quantity of N-[2-(acetamidomethyl)mercaptopropionyl]glycylN'-(2-aminoethyl)homocysteinamide (78) is used in place of the amine 8.Obtained in this manner is Compound 79 of Scheme 8.

EXAMPLE 17

1-{{2',3'-bis-{N-[(2"-mercapto-2"-methyl)propyl]amino}propyl}}-3-[(methyoxyimino)methyl]pyridiniumchloride, i.e. Compound 10 (0.17 mmol), is dissolved in 1.0 mL ofabsolute ethanol and 1.0 mL of 1N NaOH. A 1.0 mL generator eluant of^(99m) TcO₄ - (5 to 50 milliCuries) in saline is added. Then, 0.5 mL ofdithionite solution, prepared by dissolving 336 mg of Na₂ S₂ O₄ per mLof 1.0 NaOH, is added and the mixture heated sufficiently to reduce boththe technetium and the pyridinium salt and to form the complex betweenthe dihydropyridine-containing ligand and the oxotechnate-99m ion. Thecomplex so prepared, i.e. Complex 12 of Scheme 1, is buffered by theaddition of 1.0 mL of 1N NaCl and 4.0 mL of 0.1 mL of NaH₂ PO₄, pH 4.5buffer.

EXAMPLE 18

The generator procedure of Example 17 can be repeated to convertCompound 20 to Complex 22; Compound 28 to Complex 30; Compound 36 toComplex 38; Compound 46 to Complex 48; Compound 50 to Complex 52;Compound 61 to Complex 63; Compound 71 to Complex 73; Compound 79 toComplex 81; Compound 89 to Complex 91; Compound 99 to Complex 101;Compound 110 to Complex 112; Compound 118 to Complex 121; and so forth.

EXAMPLE 19

To a slurry of 11 g of lithium aluminum hydride in 300 mL of drytetrahydrofuran is added dropwise, over a 2 hour period and under anargon atmosphere, 13 g of the amide 6 in 150 mL of dry tetrahydrofuran.After the addition is complete, the reaction mixture is heated at refluxfor 30 hours, then quenched with saturated Na-K tartrate solution.Treatment with 3N hydrochloric acid and then with saturated sodiumcarbonate solution, followed by filtration and extraction of thefiltrate with dichloromethane affords an organic solution which is driedover magnesium sulfate. Removal of the solvent affords the desiredamine, Compound 8 of Scheme 1, as a viscous oil.

A sample of the free amine thus obtained is dissolved in diethyl etherand hydrogen chloride gas is added. The white powder which separates isremoved by filtration and purified from ethanol/water to give thecorresponding hydrochloride salt melting at 225°-228° C. ¹ H NMR (D₂ O)δ 3.3-4.2(m,9H,HCl,NH₂ CH₂, --HCl NHCH₂), 1.5[m,12H,C(CH₃)₂ ]. Anal.Calcd. for C₁₁ H₃₀ Cl₃ N₃ S₂. H₂ O: C.33.63; H.8.21; N.10.69; Cl,27.07;S.16.32. Found: C,33.93; H,7.94; N,10.60; Cl,27.05; S,16.25.

EXAMPLE 20

A mixture of 1 g of the amine -8, 75 mL of acetone and a catalyticamount of p-toluenesulfonic acid is heated at reflux for 24 hours. Thesolvent is removed by rotary evaporation and the residue is taken up inchloroform and treated successively with saturated aqueous sodiumbicarbonate solution, aqueous sodium hydroxide solution (10%) andsaturated aqueous sodium chloride solution. The solution is dried overmagnesium sulfate. Removal of the solvent leaves a viscous mass. Thinlayer chromatography (CHCl_(3/) methanol, 2:1) indicates two majorcomponents having Rf values of 0.13 and 0.73. The component with thelower R_(f) value shows a positive ninhydrin test, confirming that it isthe desired primary amine 8a while the component with the higher R_(f)value is negative. ¹ H NMR of the Rf 0.73 component (CDCl₃): δ 2.9, 2.5,1.3-1.5. ¹ H NMR of the R_(f) 0.13 component (CDCl₃): δ 3.0, 2.8, 2.3,1.2-1.7. Obtained in this manner is the desired tisthiazolidine primaryamine, Compound 8a of Scheme 1.

EXAMPLE 21

Reaction of the bisthiazolidine primary amine 8a with the Zincke reagent9 according to the procedure of Example 8 affords the correspondingbisthiazolidine quaternary. i.e. Compound 10a of Scheme 1, which canthen be de-protected. e.g. by reaction with mercuric chloride, followedby treatment with hydrogen sulfide, to give the unprotected quaternary.Compound 10 of Scheme 1.

EXAMPLE 22

Reaction of the bisthiazolidine primary amine 8a with the Zincke reagent69 according to the proCedure of Example 8 affords the correspondingbisthiazolidine quaternary. i.e. Compound 10b of Scheme 1; removal ofthe protecting groups. e.g. by successive treatments with HgCl₂ and H₂S, affords the corresponding unprotected quaternary, i.e. Compound 10cof Scheme 1.

EXAMPLE 23

A solution of 7 g (3 mmol) of the ester 5 (prepared, for example, asdescribed in Example 3) in 50 mL of dry tetrahydrofuran is addeddropwise over a period of 1 hour to 1.8 g (47 mmol) of lithium aluminumhydride in 200 mL of dry tetrahydrofuran. The mixture is heated atreflux for 16 hours, after which time the reaction is quenched withK--Na tartrate solution. The organic phase is dried over sodium sulfate.Removal of the solvent leaves a yellow viscous mass. Yield 4 g (65%) ofthe desired alcohol, Compound 18a of Scheme 2. ¹ H NMR (CDCl₃) δ2.2-2.8, 3.5, 2.3, 1.5.

EXAMPLE 24

A solution of 2 g of PBr₃ is added at 0° C. to 1 g of the alcohol 18a.The mixture is heated at reflux for 30 minutes, then treated withsaturated aqueous sodium bicarbonate solution and extracted withchloroform. The chloroform extract is dried over sodium sulfate. Removalof the solvent in vacuo left the corresponding bromo compound. i.e.Compound 19 of Scheme 2. as a clear viscous mass.

EXAMPLE 25

Following the general procedure of Example 20, but substituting anequivalent quantity of the alcohol 18a in place of the amine 8, affordsthe bisthiazolidine alcohol. Compound 18b of Scheme 2.

EXAMPLE 26

Reaction of the bisthiazolidine alcohol 18b with PBr₃ according to theprocedure of Example 24 affords the corresponding bromo compound, i.e.Compound 19a of Scheme 2.

EXAMPLE 27

A solution of 17 mL of 2N lithium borohydride in tetrahydrofuran isadded to 300 mL of dry tetrahydrofuran under an argon atmosphere. Tothat solution are added 10 g (0.035 mol) of the ester 40 in 100 mL ofdry tetrahydrofuran. The resultant cloudy solution is heated at refluxfor 1.5 hours. The reaction is quenched with water and the organic phaseis washed with saturated aqueous sodium chloride solution and dried overmagnesium sulfate. Removal of the solvent leaves as a white powder whichis very soluble in water, the corresponding primary alcohol, Compound 32of Scheme 4. Yield 2 g (24%); melting point 85°-90° C.; ¹ H NMR(acetone-d₆) δ 7-8, 4.15, 3.3-4.0.

EXAMPLE 28

To 1 g of the alcohol 32 in 40 mL of dry ethanol is added a solution ofsodium thiobenzoate prepared from 0.2 g of sodium in 10 mL of ethanoland 1.26 g of thiobenzoic acid in 5 mL of ethanol. The reaction mixtureis stirred at room temperature for 10 minutes, then is heated at 45° C.for an additional 10 minutes, The mixture becomes very thick anddifficult to stir and a yellow product separates. The product, Compound33 of Scheme 4, is removed by filtration and washed with water. Yield1.2 g, melting point 151°-152° C.; ¹ H NMR (DMSO-d₆ /acetone-d₆) δ7.4-8.3. 3.85. 3.1-3.6.

EXAMPLE 29

To 50 mL of dichloromethane are added 2 9 (4.5 mmol) of the alcohol 33and 0.35 g (4.5 mmol) of dry pyridine. The solution is cooled and 0.8 g(6.8 mmol) of thionyl chloride in 5 mL of dichloromethane is addeddropwise over a ten minute period. The solution is allowed to stirovernight at room temperature. Then an additional 50 mL ofdichloromethane is added and the solution is washed successively with 2Nhydrochloric acid, saturated sodium bicarbonate solution and water.Drying over magnesium sulfate and removal of the solvent left a yellowsolid having an R_(f) (CH₂ Cl₂ /acetone) of 0.47. Yield 1.7 g (81.6%) ofthe chloro derivative, Compound 34a of Scheme 4, which melts at129°-131° C.

Chelating agent precursors, chelating agents and radiopharmaceuticalswithin the purview of the present invention can also be prepared basedon the bifunctional chelating agents of Yokoyama et al U.S. Pat. No.4,287,362. Thus, for example, Yokoyama et al's chelating agents of theformula ##STR78## wherein R¹, R₂, R₃ and R₄ are each H or C₁ -C₃ alkylcan be first converted to the corresponding esters (e.g. replacing--COOH with --COOC₂ H₅), which can then be reduced to the correspondingalcohols (replacing --COOC with --CH₂ OH). which can then be convertedto the corresponding --CH₂ Br or --CH₂ Cl derivatives, which can in turnbe reacted with the selected pyridine compound of the formula ##STR79##thus replacing the halogen atom to afford quaternary salt of formula (I)herein. Other process variations will be apparent from the many reactionschemes depicted hereinabove.

Another bifunctional chelating agent which can be readily converted tothe redox system-containing chelating agent precursors, chelating agentsand radiopharmaceuticals of this invention is a compound of the formula##STR80## which is also known as amino DTS and which is described in theliterature, e.g. in Jap. J. Nucl. Med. 19, 610 (1982). Amino DTS can bereadily converted to the derivatives of the present invention byreacting it with a Zincke reagent of the formula ##STR81## wherein##STR82## is as defined with formula (I) hereinabove above to afford thecorresponding precursor of formula (I), which can then be utilized asgenerally described herein to prepare the corresponding compound offormula (II) and radiopharmaceuticals of formulas (III) and (IV). See,for example. Scheme 14 below.

Yet another group of known chelating agents which is particularlywell-suited for conversion to the redox system-containing chelatingagent precursors, chelating agents and radiopharmaceuticals of thepresent invention can be represented by the formula ##STR83## whereinR₁, R₂, R₃ and R₄ are each H or C₁ -C₃ alkyl and n' is an integer of 0to 3. See, for example. Yokoyama et al U.S. Pat. No. 4,511,550 andAustralian Patent No. 533,722. An especially preferred chelating agentencompassed by this group is known as amino-PTS, or AEPM, and has thestructure ##STR84## Amino-PTS can be converted to the derivatives of thepresent invention via a Zincke reagent, as described supra in connectionwith amino-DTS. See, for example, Scheme 15 below. The exact structureof the resultant technetium complex 136 has not been determined; it ispossible that the C═N and C═S bonds are also reduced during one of thereduction steps. One possible structure for 136 is as follows: ##STR85##(A similar structure could be depicted for complex 131 of Scheme 14.)

An alternate route to derivatives of amino PTS, amino DTS and the likeis depicted in Schemes 16 and 17 below. This route can begin byutilizing known, commercially available pyrylium salts. e.g. Compound138, to convert the primary amino group of amino PTS, amino DTS or thelike into a pyridinium intermediate. The resultant pyridiniumintermediate (e.g. 140 or 143) can then undergo nucleophilicdisplacement to afford the corresponding halo compound (e.g. 141 or144). The halo derivative can then be reacted the selected pyridinecompound of the formula ##STR86## to afford the corresponding quaternarysalt of (I) herein, which can be converted to the instant derivatives offormulas (II), (III and (IV) as already described hereinabove. ##STR87##

Fritzberg U.S. Pat. No. 4,444,690 describes an interesting series of2,3-bis(mercaptoalkanoamido)alkanoic acid chelating agents of thegeneral formula ##STR88## wherein X is H or --COOH, and R and R' are Hor lower alkyl, and water-soluble salts thereof, used to prepare thecorresponding radiopharmaceuticals of the formula ##STR89## wherein X isH or --COOH, and R and R' are H or lower alkyl. The Fritzberg chelatingagents are prepared from the corresponding 2,3-diaminoalkanoic acids byesterification with a lower alkanol containing dry HCl, followed bytreating the resultant alkyl ester with a chloroalkanoyl chloride toform the bis(chloroalkanoamide)ester, followed by treating that esterwith ##STR90## followed by alkaline hydrolysis of the resultant2,3-bis(benzoylmercaptoalkanoamido)alkanoic acid ester to produce the2,3-bis(mercapto-alkanoamido)alkanoic acid chelating agent. Preparationof an analog from 3,4-diaminobenzoic acid is also disclosed byFritzberg. Many of Fritzberg's synthetic steps can be adapted to producethe formula (I) derivatives of this invention in which, in place of the--COOH group in Fritzberg's chelating agent, there is a ##STR91## orlike group, wherein and X⁻ are as defined with formula (I) hereinabove.See, for example, Schemes 4, 5, 6 and 11 hereinabove.

Suitable nontoxic pharmaceutically acceptable diluents or vehicles foruse with the present complexes of formula (III) will be apparent tothose skilled in this art. See, for example, Remington's PharmaceuticalSciences, 4th Edition (1970). Obviously, the choice of suitable diluentsor vehicles will depend upon the exact nature of the particular dosageform selected.

The dosage ranges for administration of the complexes according to thisinvention will vary with the size and species of the subject, theobjective for which the complex is administered, the particular dosageform employed, and the like, as discussed below. The quantity of givendosage form needed to deliver the desired dose of theradiopharmaceutical, of course, depends upon the concentration of thecomplex in any given pharmaceutical composition/dosage form thereof andthe radioactivity thereof.

By way of example only, a 5-50 mg/kg dose of formula (III)radiopharmaceutical, injected into the tail vein or carotid vein ofrats, due to the "lock in" mechanism will exhibit a very significantdifference between brain and peripheral levels of radioactivity, withconsequent ready radioimaging of the brain; imaging at approximately 60to 90 minutes after administration will be most effective, since it willtake advantage of this brain/peripheral differential.

The instant radiopharmaceuticals are generally administeredintravenously. Sustained release administration, typically by slowintravenous infusion, will further enhance the site-specificity of theinstant redox system. The rate of release of the formula (III)radiopharmaceutical from the sustained release system should becomparable to the rate of in vivo oxidation of the dihydro form (III) tothe quaternary form (IV) in order to achieve the greatest degree ofenhancement of specificity.

In a further aspect, the present invention also provides a process forthe manufacture of a diagnostic agent for the visualization of an organsuch as the brain. To that end, the blood-brain barrier- penetratingform, formula (III), is admixed with an aqueous buffer medium having apH value of about 4 to about 8, preferably of about 6.5 to about 7.5, inan effective radioimaging amount.

Preparation of the radiopharmaceutical can be carried out in thehospital or like location where the patient is found in order tominimize losses of radioactivity caused by the decay of radioactivemetal. Inasmuch as the preparation for visualization is injectable, itmust be sterile and pyrogen-free; preferably, it is also isotonic. Tothis end, a so-called labeling kit can be provided that permits asimple, rapid and safe labeling of the solution to be injected with theradioactive metal e.g., technetium-99m. Such kits are especiallydesirable when a short-lived radioisotope such as technetium-99m isused.

The kit includes a collecting vial for receiving and/or containing anaqueous medium in which the complexing reaction can be effected.Additionally, the kit includes the chelating agent of formula (II) orchelating agent precursor of formula (I) and a pharmacologicallyacceptable reducing agent for reducing the radioactive element to anappropriate oxidation state for complexing with the chelating agent [andalso for reducing the pyridinium moiety to the correspondingdihydropyridine form, when a chelating agent precursor of formula (I) ispresent].

In the case of technetium-99m, the radioactive element is received froma radionuclide generator as an aqueous pertechnetate (TcO₄ ⁻) solutionsuch as an eluate in isotonic saline, as is well-known in the art. Theamount of Tc-99m required to produce a quantity of formula (III)radiopharmaceutical sufficient for diagnostic purposes is generally from0.01 milliCurie (mCi) to about 500 mCi per mL of 99m-pertechnetatesolution. The reducing agent for the pertechnetate can be a thiosulfateor dithionite if the reducing reaction is to be carried out in a basicmedium, or a tin (II) salt such as SnCl₂ if the reducing reaction is tobe carried out in an acid medium.

A kit for preparing an injectable radiopharmaceutical, e.g., forcomplexing an organ-specific agent labeled with a radioactive metal,includes, in separate containers: (1) a biologically compatible, sterileaqueous medium suitable for complex formation with a radioactive metal,(2) a dihydropyridine←→pyridinium salt redox system-containingcomplexing agent of formula (I) or (II) compatible therewith, and (3) apharmaceutically acceptable reducing agent for the radioactive metal.

The dihydropyridine←→pyridinium salt redox moiety may be present in thekit either in its oxidized or its reduced state, as desired. Thereducing agent for the radioactive metal can be selected to reduce alsothe oxidized form of the redox moiety, if present, as the radioactivemetal is reduced to form the complex preparatory to injection of theradiopharmaceutical into a test animal or a patient. In a preferredembodiment of this invention, a reducing agent capable of reducing boththe oxidized form of the redox moiety and the radioactive metal ischosen and the chelating agent precursor of formula (I) is present inthe kit. In an especially preferred embodiment, the kit comprises, inseparate containers (preferebly aspetically and hermetically sealedvials of approximately 5-25 mL volume), (1) a biologically compatible,sterile aqueous medium, (2) a chelating agent precursor of formula (I).and (3) a pharmacologically acceptable reducing agent capable ofreducing the chelating agent precursor of formula (I) to a chelatingagent of formula (II) and also capable of reducing the radioactive metalto an oxidation state in which it is capable of complexing with theformula (II) chelating agent to form a radiopharmaceutical of formula(III). Most preferably, the reduCing agent is sodium dithionite; alsomost preferably, the radioactive metal is technetium. The dithionitereduction is preferably carried out in basic medium; this may beaccomplished by providing that the aqueous medium (1) above is of basicpH, or by adding an appropriate base (e.g. NaOH, Na₂ CO₃) when combiningthe kit components and the pertechnetate solution. As yet anotheralternative, the kit could comprise only two separate components: (1)the biologically compatible, sterile aqueous medium of essentiallyneutral pH containing the chelating agent precursor of formula (1); and(2) the reducing agent e.g. sodium dithionite or (2) the reducing agenttogether with the base, e.g. sodium dithionite and sodium carbonate.

Radioactive metal ions are typically not provided with the kit due tothe relatively short half-lives of commonly utilized radionuclides.Rather, the radionuclide is provided separately as described earlier andadmixed with the components of the kit shortly before use, as is knownfor other radiopharmaceutical delivery systems. In the case oftechnetium-99m, the pertechnetate solution and the basic aqueous mediummay be first combined and then heated. e.g. from 40° to 95° C. for 10 to20 minutes. in the presence of the reducing agent, then cooled to aboutroom temperature or below prior to addition of the formula (I)precursor. In this instance, the technetium will be reduced prior toreduction of the quaternary moiety to the corresponding dihydro form, inwhich case a substantial portion of the quaternary salt (I) will likelychelate with the reduced technetium to form the quaternary complex (IV)in the reaction mixture as an intermediate to the dihydro complex (III),rather than the quaternary salt (I) being first converted to the dihydrochelating agent (II) and then to the dihydro complex (III).Alternatively, if only minimal or no heating is done, the precursor maybe present in the initial mixture made from the kit, and it is likely inthis instance that the formula (I) quaternary will be first reduced tothe formula (II) dihydro, which will then chelate with the reducedtechnetium to form the complex (III). If the mixture is mildly basic,e.g. pH 8 to 9, it may be administered as is, after the reduction andchelation have occurred to form the formula (III) radiopharmaceutical,or the pH may be adjusted to about 7. If the mixture is more stronglybasic, e.g. pH 13, it is generally desirable to adjust the pH to aslightly alkaline or neutral value.

Whatever the exact configuration of the kit, it is preferable for it tocontain excess chelating agent precursor (I) or chelating agent (II)with respect to the radionuclide to be complexed therewith, e.g. a 1:2molar excess. The reducing agent is present in a large excess withrespect to the chelating agent precursor (I), e.g. 1:5 to 1:10. When thechelating agent (II) rather than the precursor (I) is present, then thereducing agent is preferably present in a slight excess with respect tothe radionuclide.

To effect visualization, the diagnostic agent is administered to apatient, typically intravenously, with or without further dilution by acarrier vehicle such as physiological saline, phosphate-buffered saline,plasma, or the like. Generally, the unit dose to be administered has aradioactivity of about 0.01 milliCurie (mCi) to about 100 milliCuries,preferably about 1 mCi to about 20 mCi. The solution to be injected intoan adult patient per unit dosage is about 0.01 milliliter (mL) to about1 milliliter.

After intravenous administration, imaging of the organ in vivo can takeplace after a few minutes. If desired, imaging can also take place hoursafter the injection, depending upon the half-life of the radioactivematerial that has been introduced into the patient and upon the amountof such material introduced. Preferably, imaging takes place 60 to 90minutes after intravenous administration.

Any conventional method of imaging for diagnostic purposes can beutilized when practicing the present invention.

In summary, then, in its broadest aspects the present invention can beseen to provide compositions of matter comprising: (1) the residue of achelating agent having at least one primary, secondary or tertiary aminofunctional group, said functional group being not essential for thecomplexing properties of said chelating agent, said residue beingcharacterized by the absence of at least one of said primary, secondaryor tertiary amino functional groups, said chelating agent being either(a) capable of chelating with a metallic radionuclide or (b) chelatedwith a metallic radionuclide; and (2) a dihydropyridine←→pyridinium saltredox system, which in its oxidized form comprises a radical of theformula ##STR92## wherein, n, p, q, and R are as defined with formula(I) hereinabove, and which in its reduced form comprises a radical ofthe formula ##STR93## wherein n, p, q, m and R are as defined withformula (II) hereinabove; said redox system being directly attached tosaid chelating agent residue, the ring nitrogen atom of said redoxsystem occupying the same position relative to said chelating agentresidue as the position occupied by said primary, secondary or tertiaryamino functional group in said chelating agent.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims.

What we claim is:
 1. A compound having the structural formula ##STR94##wherein the dotted line indicates the presence of a double bond ineither the 4 or 5 position of the dihydropyridine ring; n is zero, oneor two; and each R is independently selected from the group consistingof halo, C₁ -C₇ alkyl, C₁ -C₇ alkoxy, C₂ -C₈ alkoxycarbonyl, C₂ -C₈alkanoyloxy, C₁ -C₇ haloalkyl, C₁ -C₇ alkylthio, C₁ -C₇ alkylsulfinyl,C₁ -C₇ alkylsulfonyl, --CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl, and--CONR'R" wherein R' and R", which can be the same or different, areeach H or C₁ -C₇ alkyl.
 2. A compound as defined by claim 1, wherein nis one.
 3. A compound as defined by claim 2, wherein R is located in the3 position of the dihydropyridine ring.
 4. A compound as defined byclaim 2, wherein R is --CH═NOR'" wherein R'"is H or C₁ -C₇ alkyl, orwherein R is --CONR'R" wherein R' and R", which can be the same ordifferent, are each H or C₁ -C₇ alkyl.
 5. A compound as defined by claim3, wherein R is --CH═NOR'" wherein R'" is H or C₁ -C₇ alkyl, or whereinR is --CONR'R" wherein R' and R", which can be the same or different,are each H or C₁ -C₇ alkyl.
 6. A compound as defined by claim 5, whereinthe dotted line indicates the presence of a double bond in the 4position of the dihydropyridine ring.
 7. A compound having thestructural formula ##STR95##